PROJECT SUMMARY/ABSTRACT Calsequestrin-2 (Casq2) is a high capacity, low affinity calcium (Ca) binding protein located in the junctional sarcoplasmic reticulum (SR) of cardiac myocytes. As the major SR Ca buffer, Casq2 interacts with the ryanodine receptor (RyR2), a Ca release channel, to regulate the amount of Ca that is released during the excitation- contraction (EC) coupling cycle, a process that couples electrical activation to mechanical force (i.e. a heartbeat). Alterations in EC coupling can cause both contractile dysfunction and cardiac arrhythmias. Reduction or loss of Casq2 due to mutations causes a severe genetic arrhythmia syndrome known as catecholaminergic polymorphic ventricular tachycardia (CPVT). Genetic variants of Casq2 have also been associated with sudden cardiac death and heart failure in patients with coronary artery disease, while overexpression of Casq2 causes hypertrophy and heart failure in mice. Casq2-linked CPVT is usually autosomal-recessive, with mutations resulting in either a severe reduction or complete loss of Casq2 protein. As a result, SR Ca buffering is reduced, which leads to spontaneous Ca release and arrhythmias. In 2016, a genetic analysis conducted in a family that had an autosomal dominant inheritance of CPVT uncovered a novel missense mutation (K180R) within Casq2. This was the first autosomal dominant mutation found in Casq2. Initial studies in heterozygous K180R knock-in mice demonstrate that protein levels of Casq2 are normal but mice exhibit CPVT when stressed. This suggests that Casq2-K180R causes CPVT by a different mechanism than previously reported autosomal-recessive Casq2 mutations. I hypothesize that Casq2-K180R causes CPVT by disrupting its ability to regulate RyR2 Ca release channels, either directly or by altering SR Ca buffering, leading to spontaneous Ca release. To test this, I plan to use both mouse and human pluripotent stem cell models. Recent studies have shown that cardiomyocytes (CM) differentiated from human induced pluripotent stem cells (hiPSCs) can model CPVT and be used to screen potential therapeutics. Utilizing the K180R mouse and hiPSC models I already generated, the aims of this project are to determine how K180R affects SR calcium handling and to investigate how K180R affects Casq2 Ca binding, localization, and polymerization. I will investigate how CMs are effected at the physiological and cellular levels and determine how Casq2 is altered at the protein level. This project will provide new insight into the role of Casq2 during the EC coupling cycle, the functional interaction between Casq2 and RyR2, the termination of SR Ca release, and how Casq2 variants could lead to cardiac arrhythmias and/or heart failure. This improved understanding of Casq2 could lead to better treatment strategies for patients suffering from Casq2-dependent cardiac disorders.